Do "RED" mutants of GFP exist?

In an e-mail and bulletin sent out yesterday, Dr. C.S. Prakash wrote,
"By substituting amino acids in the chromophore, proteins which yield blue
and red fluorescence have been developed (Heim, et al 1994 PNAS
91:12501-12504; Delagrave, et al. 1995 Bio/Technology 13:151-154)."
The claim that a GFP mutant with red fluorescence currently exists seems
one of the more durable misconceptions about GFP. The actual title of the
Delagrave et al paper is "Red-Shifted Excitation Mutants of the Green
Fluorescent Protein". As stated within that paper, these mutants have their
emission peaks near 505 nm, which is still green, NOT red. Fig. 2 of that
paper, which might on cursory inspection seem to show red colonies, is a
pseudocolor image obtained with two different EXCITATION wavelengths. The
phrase "red-shifted" is a spectroscopic term merely describing a shift to
longer wavelengths. Unfortunately it seems to have confused many
biologists, who want red and easily forget the words "-shifted excitation".
To my knowledge the GFP mutant with the longest wavelengths is P4-1
(Heim & Tsien (1996) Current Biology 6: 178-182), whose excitation and
emission maxima are 504 and 514 nm respectively. 514 nm is still not red,
not even yellow. This paper also describes mutants that genuinely emit
blue (e.g. 445 nm), improved beyond the 1994 versions cited by Dr.
Prakash. If anyone out there does have a truly red mutant of GFP, i.e. with
an emission peak > 600 nm, many of us would like to hear about it.
Roger Y. Tsien
Howard Hughes Medical Institute and Univ. California, San Diego
Tel: (+1)619-534-4891, fax (+1)619-534-5270